J. Org. Chem., Vol. 36, No. 18, 1971 2609
KE~o-ti-endo-.( 2-IM1DAZOLYL)BICYCLO[2.2.2]OCTANES Typical experiments are summarized in Tables 1 and 11. The results of a series of experiments are listed in Table 111.
Registry No.-( A)-$ 30256-03-2; ( -)-3,
30318-
67-3; (*)-4, 30256-04-3; ( * ) - 5 , 30247-99-5; (+)-5, 30248-00-1; ( * ) - 6 , 30275-69-5; (+)-6, 30248-01-2; (*) inactive polymer, 30228-77-4; (+) inactive polymer, 30228-78-5.
Synthesis of 2- and 3-I~eto-5-endo-(2-imidazolyl)bicyclo[2.2.2]octane ARTHURF. WAGNER,* PAUL E. WITTREICH,BYRONH. ARISON,AND LEWISH. SARETT Merck Sharp & Dohme Research Laboratories, Division of Merck & Co., Inc., Rahway, New Jersey 07066 Received August 31, 1970 On the basis of a concept of attributes that make compounds attractive candidates for biological screening, %keto- and 3-ketDo-5-endo-(2-imidazolyl)bicyclo [2.2.2] octane were synthesized on a relatively large scale for conversion to a series of corresponding 2- or 3-substituted analogs.
I n order to raise the probability of finding biological activity among candidates for pharmacometric screening, a family of compounds with structural features that should a prior% enhance their effectiveness was synthesized. Three features included in this particular series were (1) a rigid bicyclic framework for favorable entropy of binding to a receptor, (2) an imidazole nucleus, attractive for its bifunctional nature and participation at active siteei of enzymes, and (3) a second functional group chosen from those found among naturally occurring compounds. Although the biological activity of the group was disappointing, certain aspects of this synthetic organic chemistry are worthy of note. On the basis of the criteria outlined above, 5-endo(2-imidazolyl) bicyclo [2.2.2]oct-2-ene (4) was selected as an appropriate starting compound. The most logical way to synthesize 4 is by a Diels-Alder condensation between cyclohexadiene-1,3 and a 2-vinylimidazole, but this method was soon abandoned in favor of a stepwise synthesis starting from 5-endo-cyanobicyclo [2.2.2]oct-2-ene. The target compound 4 was synthesized starting with the conversion of 5-endo-cyanobicyclo [2.2.2]oct2-ene2 (1) to 5-endo-carbiminomethoxybicyclo [2.2.2]oct-Zene hydrochloride (2) on treatment with methanol and hydrogen chloride in ether solution. Reaction of the imino ether 2 with (3-aminoacetaldehyde diethyl acetal yielded N-(@,~-diethoxyethyl)-5-endo-carbamidinobicyclo [2.2.2]oct-2-ene hydrochloride (3), which at 120" in glacial acetic acid-acetic anhydride gave 5endo-(2-imidazolyl) bicyclo [2.2.2Ioct-2-ene (4). A critical step in the scheme required the introduction at an appropriate position on the bicyclic framework of a carbonyl group which could then be converted into a variety of functional groups. The unsaturation a t the 2,3 position of' the bicyclic skeleton was a logical point to attack, but more difficulty was encountered than anticipated in applying typical reactions for this trans(1) T h e relatively poor dienophilic nature of vinylimidazoles and their propensity to polymerize at relatively low temperature did not augur well for a successful Diels-Alder condensation. I n addition, considerable difficulty was justifiably anticipated in the prepartion of certain 2-vinylimidazoles on a reasonably large scale. Nevertheless, 1-benzyl-2-vinylimidaeole was synthesized from I-benzyl-2-lithioimidazole by reaction with acetaldehyde and dehydration over fused KHSOs a t 251-230' (25-45 mm). The distilled product (ea. 60% pure) did not undergo Diels-Alder condensation with cyclohexadiene-1,3 a t temperatures up t o 170' or a t pressures u p t o 138,000 psi. I n a parallel attempt, methyl p-(4-imidazolyl)acrylate was prepared from L-histidine and heated with cyclohexadiene-l,3 at temperatures up t o 210° without auccess. (2) K. Alder, H. Heimbach, and R . Reubke, Chem. B e y . , Si, 1516 (1958).
formation. The imidazole moiety appeared to be largely responsible for the difficulty by either preventing reaction at the 2 or 3 position or by being susceptible to attack by the reagent. For example, in a variety of hydroboration studies, stable aminoborane derivatives were isolated and the unsaturated moiety was intact. I n other instances involving mild oxidizing agents, the imidazole ring was attacked. Facile conversion of the unsaturated compound 4 to 2-keto-5-endo-(2-imidazolyl)bicyclo[2.2.2]octane (5) was achieved by palladium chloride oxidation in aqueous medium.3 The reaction is usually accomplished by using catalytic quantities of palladium chloride in the presence of large amounts of cupric chloride and an air stream. The propensity of the keto imidazole derivative 5 to chelate with copper, however, made it prudent to use palladium chloride in a t least stoichiometric amount and eliminate the cupric salt.
I
1
OCH, 2
I
C=NH.HCl
I HNCH,CH(OC,H,),
NANH
u
4
u
5
3
The location of the carbonyl group in 5 could not be established on theoretical grounds and was not immediately obvious from nmr spectra data in CDC13. In the absence of model compounds, chemical shifts could not be exploited with assurance, since the positions of protons adjacent to the ketone would be similar in both the 2 and 3 isomers. Furthermore, any difference in multiplicity between the bridgehead protons could not be used as an approach to structure assignment because of the coincidence of the CI and C b proton peaks in severgl solvents. These difficulties were overcome by taking advantage of the long-range coupling (3) For a review see J. Smidt, W. Hafner, R. Jira, R. Sieber, J. Sedlmeier, and A. Sabel, Angew. Chem., Int. Ed. En&, 1, 80 (1962).
2610
J. Org. Chem., Vol. 36, No. 18, 1971
often observed between exo protons separated by four single bonds.4 In the bicyclo [2.2.2]octane system, this would be reflected by coupling between the c6 and Cs exo protons. The detection of such interaction, most readily determined from a critical examination of the c6 resonance, would therefore exclude the Casite for the ketone. I n pyridine, the C5 proton resonance is composed of 11 peaks. This is consistent with an overall multiplicity of 16 lines arising from coupling constants of 10.7, 6.0, 2.0, and 2.0 Hz. The observed pattern results from the coincidence of lines 2-3, 6-7, 8-9, 10-11, and 14-15. Since the three vicinal protons can account for a maximum of eight lines, it follows that the C5 proton is involved in long-range coupling with a fourth proton, presumably the exo proton a t C3. Compound 5 was therefore assigned the 2-keto structure. This line of reasoning was later confirmed when it was shown that the C5 proton resonance in the 3-keto isomer, prepared by an unambiguous synthesis, was composed of eight lines. For the synthesis of the 3-keto analog 11, bicyclo[2.2.2]oct-2-ene-5-carboxylic acid5served as the starting material. Treatment of this acid with 30% sulfuric acid at 110" for 1 hr6 gave excellent yields of 3-endohydroxy- 5 - endo- carboxybicyclo [2.2.2]octane lactone (6),which on ammonolysis yielded 3-endo-hydroxy-5endo-carbamylbicyclo [2.2.2]octane (7). Treatment of the hydroxy analog 7 with chromic acid in acetic acid gave the corresponding keto derivative 8, which on dehydration with p-toluenesulfonyl chloride in pyridine
& & --t
0-c=o
HO
6
CONHz
7
8
9
10
yielded 3-keto-5-endo-cyanobicyclo [ 2.2.2 ]octane (9). The cyano derivative 9 was converted to 3-keto-5endo-(2-imidazolyl)bicyclo[2.2.2]octane (1 1) by way of the imino ether 10. (4) F. A. L. Anet, Can. J . Chem., 89, 789 (1961): T. F. Flautt and W. F. Erman, J . Amer. Chem. Soc., 86, 3212 (1963); J. Meinwald and Y. Meinwald, ibid., 86, 2514 (1963): K. Tori, Y.Takano, and K. Kitahonoki, Chem. Ber., 97, 2798 (1964). ( 5 ) R. Seka and 0. Tramposh, ibid., 76, 1379 (1942). (6) These conditions are superior t o those reported by K. Alder and G. Stein, Justus Liebigs Ann. Chem., 614, 197 (19341, or W. R. Boehme, E. Schipper, W. G. Scharf, and J. Nichols, J . Aner. Chem. SOC.,80, 5488 (1958).
WAGNER,WITTREICH,ARISON,AND SARETT Experimental Section' 5-endo-Carbiminomethoxybicyclo[2 .2.2] oct-2-ene Hydrochloride (2).-A solution of 2 g (62 mmol) of BleOH and 8.3 g (62 oct-2-ene2 in 18 ml of anhymmol) of 5-endo-cyanobicyclo[2.2.2] drous Et20 was cooled, and 2.3 g of anhydrous HC1 in EtzO was added. After being allowed to stand overnight a t S o , the mixture was filtered, yielding 6.6 g of 2 , mp 165-167'. Anal. Calcd for CloHlsClNO: C, 59.55; H , 8.00; C1, 17.58; N, 6.95, Found: C, 59.86; H , 8.16; C1, 17.46; N, 7.17. N-(P,p-Diethoxyethy1)-5-endo-carbamidinobicyclo [ Z .2.2] oct-2ene Hydrochloride (3).-A suspension of 40 g (0.2 mol) of 2 in 87 ml of MeOH was warmed to 30" and 27.4 g (0.21 mol) of P-aminoacetaldehyde diethyl acetal was added rapidly. The temperature of the suspension rose to 51", and the mixture became clear. After l hr, the solution was Concentrated under reduced pressure, yielding 62.3 g of 3. 5-endo-(2-Imidazolyl)bicycloj2.2.21 oct-2-ene (4).-A solution of 62.3 g (0.2 mol) of 3 in 260 ml of AcOH and 111 ml of Ac20 was heated a t 120' for 2 hr. The reaction mixture was concentrated under reduced pressure, and the product was dissolved in 270 mi of 2.5 iV HCl. The acidic solution was washed with E t 2 0 , made alkaline with 50% KOH, and extracted with CHC1,. The CHCh extract was washed with HzO, dried over MgS04, filtered, and concentrated under reduced pressure. The residue was taken up in a small volume of CHCL and adsorbed on 500 g of silica gel packed in CHtCl2. The product was eluted with 0.5% BIeOH in CHzC12 and recrystallized from 100 ml of C6H6petroleum ether (bp 30-60') yielding 20.95 g of 4, mp 165-167'. Anal. Calcd for CllH14Nz: C, 75.82; H , 8.10; N, 16.08. Found: C, 76.32; H , 7.83; N , 15.83. Repeated crystallization of the product failed to improve the elemental analysis. Accordingly, 13.5 g of 4 was treated with 6 ml of methyl chloroformate and 32 g of KzC0, in 400 ml of MezCO. The mixture was filtered and concentrated, and an EtzO-soluble fraction was isolated by trituration of the residue. Concentration of the Et20 solution yielded 11.8 g of the 1-carbomethoxy analog of 4, which on treatment with 250 ml of 1 N HCl yielded 7.8 g of 4. Passage of this material through 10 g of silica gel as above yielded 6.2 g of 4, mp 163-164'. Anal. Calcd for CllHl4Nz: C, 73.82; H, 8.10; N, 16.08. Found: C, 75.66; H, 7.81; N, 15.95. 2-Keto-5-endo-(2-imidazolyl)bicyclo[2.2.2]octane(5).-A mixture of 14.5 g (83 mmol) of 4, 10 g of PdC12, and 14.5 g of CuCl2. 2Hn0in 170 ml of 1 N HC1 and 900 ml of HzO was stirred and heated a t 70-80' for 1hr while air was bubbled through the solution continuously.* The hot solution was filtered and cooled, and 140 g of Na4EDTA was added. The solution was extracted five times with CHCls, and the combined extracts were dried over MgS04, filtered, and concentrated under reduced pressure, yielding an 8.2-g residue. The product was adsorbed on silica gel packed in CHzClZ, and the column was developed slowly with 0-1% MeOH in CH2C12. The product was eluted with 1-201, MeOH in CHzClz and recrystallized from CHC13-Et20, yielding 7.9 g of 5 , mp 194-196'. Anal. Calcd for CllH14N20: C, 69.44; H , 7.42; N, 14.73. Found: C,69.73; H , 7.71; N , 14.71 For a more readily purified product the following alternative procedure may be used. A mixture of 49 g of PdClz in 250 ml of 1 N HC1 was warmed to B O ' , diluted with 2250 ml of hot HzO, and heated under reflux. Next, 44 g of 4 was dissolved in 250 ml of warm 1 N HC1, and the solution was diluted with 800 ml of warm HzO. The solution of 4 was added to the refluxing PdClz mixture in the course of 0.5 hr, and the mixture was stirred under reflux for 8 hr. The mixture was filtered and the filtrate was concentrated to a 700-ml volume. The pH of the concentrate was adjusted to 8 with 3070 NHkOH and the product was extracted continuously with CHCla. Concentration of the CHCla extract yielded 39.3 g (82%) of product, which is readily purified by chromatography on silica gel (above) without prior slow development, yielding 8.8 g of starting material and 29.0 g of 5, mp 199-200'. Anal. Calcd for CllH14N20: C, 69.44; H , 7.42; N, 14.73. Found: C, 69.53; H, 7.49; N, 14.65. (7) Melting points were determined on a Koefler hot stage and are uncorrected. The ir spectra of all the compounds were consistent with the assigned structures. The nmr spectra. were obtained on Varian A-60 and HA-100 spectrometers: concentrations were generally 5-10% ( w / u ) , although a few samples were examined in the lO-ZO% range on the lower frequency instrument. (8) The CuC12.2Hg0 and air are used t o regenerate PdCli b u t complicate purification of the product. It is more convenient t o use an excess of PdCli and omit the CuCh * H i 0 and air flow.
J . Org. Chem., Vola36, N o . 18, 1971 2611
TUMOR INHIBITORS 3-endo-Hydroxy-5-endo-carboxybicyclo[2.2.2] octane Lactone (6).-The published methodsawere modified as follows. Bicyclo[2.2.2]oct-2-ene-5-carboxylic acid6b6(100 g) in 720 ml of 30% (v/v) HzS04was stirred and heated a t 110" for 1hr. The mixture was cooled, poured onto ice, and extracted with CHCls. The extract was washed with 10% NaHCOa, dried over MgSO4, filtered, and Concentrated under reduced pressure, yielding 74.3 g of 6, mp 207-208'. 3-endo-Hydroxy-5-endo-carbamylbicyclo [2.221 octane (7).-A solution of 500 mg of 6 in 10 ml of MeOH and 10 ml of liquid NHs was heated a t 110' for 12 hr. The reaction mixture\was concentrated under reduced pressure, and the residue was recrystallized from hot CHCla-petroleumether, yielding 500 mg of 7,mp 188.5189'. Anal. Calcdfor CoHlaN02: C , 63.88; H , 8.94; N, 8.28. Found: C, 64.09; H , 9.24; N , 8.50. 3-Keto-5-endo-carbamylbicyclo [2.2.2]octane @).--A solution of 28.6 g of CrOs i n 358 ml of 90% AcOH was added dropwise in the course of 2 hr to a stirred solution of 40 g of 7 in 385 ml of AcOH. After being stirred overnight, the mixture was concentrated under reduced pressure, diluted with 300 ml of HzO, and extracted continuously overnight with CHCl3. The extract was dried over MgS04, filtered, and concentrated under reduced pressure, yielding 26.3 g of crystallized product that was used in the next step. Recrystallization of a small sample of the product from R'leOH-EtzO gave 8, mp 183-184". Anal. Calcd for C9HlsNOz: C , 64.65; H , 7.84; N , 8.38. Found: C, 64.63; H , 7.93; N , 8.57. 3-Keto-5-endo-cyanobicyclo [2.2.2]octane (9).-A mixture of 1.15 g (7 mmol) of 8 and 1.45 g (7.7 mmol) of TsCl in 5 ml of pyridine was heated a t 80' for 2 hr. The mixture was diluted with 5 ml of HzO and concentrated; the residue as taken up in CHC13, and the solution was concentrated. After the residue was taken up in (3HClaand the solution was concentrated a second time, the same procedure was repeated with C&. The residue
was then triturated with C&, and the mixture was filtered. The CsH6extract was concentrated and the residue was recrystallized from CHC13-petroleum ether, yielding 0.55 g of 9, mp 145-152' (sub1 90-130"). Anal. Calcd for C9H11NO: C, 72.45; H , 7.43; N,9.39. Found: C, 72.52; H , 7.36; N , 9.44. 3-Keto-5-endo-carbiminomethoxybicyclo[2.2.2]octane Hydrochloride (lo).-A solution of 250 mg of 9 and 100 mg of MeOH in 5 ml of Et20 was cooled and saturated with anhydrous HC1. The next day, the mixture was poured into 100 ml of EtzO and filtered, yielding 220 mg of 10, mp 170-175". 3-Keto-5-endo-(2-imidazolyl)bicyclo[2.2.2] octane (1l).-A & o h tion of 107 g (0.49 mol) of 10 in 240 nil of MeOH was added dropwise in the course of 45 min to 144 g (1.08 mol) of p-aminoacetaldehye diethyl acetal a t 55-60". The mixture was stirred a t room temperature for 1 hr and concentrated under reduced pressure, yielding a 239-g residue. The product was dissolved in 1 1. of 6 A' HCl and heated under reflux for 1 hr. The solution was cooled, extracted with CHCls, made alkaline with concentrated NHIOH, and extracted with CHCls. Concentration of the latter yielded a 60-g residue that was purified by adsorption on 550 g of silica gel packed in CHzC12, elution with 47, MeOHCHzC12, and recrystallization from CH&le-Et*O, yielding 37.1 g of 11, mp 144-147'. Anal. Calcd for Cl1HI4Nz0: C, 69.44; H , 7.42; N, 14.73. Found: C, 69.63; H, 7.49; N, 14.70.
Registry No.-2, 30338-51-3; 4, 30338-52-4; 5 , 30338-53-5; 6 , 20507-79-3; 7, 30338-55-7; 8, 3033856-8; 9., 30338-57-9 ; 10, 30338-58-0; 11, 30338-59-1. Acknowledgment.-We are indebted to Rlr. A. P. Sullivan, Jr., for developing the PdClz oxidation of 4 to 5 in the absence of copper salts and to Mr. R. N. Boos and associates for elemental analyses.
Tumor Inhibitors. LXV.] Bersenogenin, Berscillogenin, and 3-Epiberscillogenin, Three New Cytotoxic Bufadienolides from Bersama abyssinica2 S. MORRISKUPCHAN,*~ J. L. RIONIOT,C. W. SIGEL,AND R. J. HEMINGWAY Department of Chemistry, University of Virginia, Charlottesville, Virginia 88901, and Department of Pharmaceutical Chemistry, University of Wisconsin, Madison, Wisconsin 63706 Received February 1, 1971 An ethanol extract of the fruits of Bersama abyssinica was found to show significant inhibitory activity against cells derived from human carcinoma of the nasopharynx carried in cell culture (KB). The isolation and structural elucidation of three new cytotoxic bufadienolidea, berscillogenin (l),3-epiberscillogenin (2), and bersenogenin (3), are reported. Mass spectrometry and elemental analysis indicated that all three compounds had a C&& molecular formula. Chemical and spectral evidence support assignment of structure 1 (16p-hydroxyscilloglaucosidin) for berscillogenin. The same enone (7)was obtained from manganese dioxide oxidation of 1 and 2, indicative that the compounds are C-3 epimers. Treatment of 3 with 80% acetic acid afforded both 1 and 2. This reaction and the nmr spectrum of 3 indicated that it is a A3-5p-hydroxyisomer of berscillogenin (1). The isolation and identification of hellebrigenin 3-acetate (4) and scilliglaucoaidin ( 5 ) are also discussed.
I n the course of a continuing search for tumor inin cell culture (KB).6 Previously, we have reported syshibitors of plant rigi in,^ we found that extracts of the tematic studies of the KB-inhibitory principles of the fruits of Bersama abyssinica Fresen. ( M e l i a n t h ~ c e a e ) ~ stem bark of B. abyssinica which led to the isolation showed significant inhibitory activity against cells deand structural elucidation of the cytotoxic principles, rived from human carcinoma of the nasopharynx carried hellebrigenin 3-acetate and hellebrigenin 3,5-diacetate,' as well as four novel naturally occurring bufadi(1) P a r t LXIV. 6. M. Kupchan, I. Ognyanov, and J. L. Moniot, Bioorg. Chem., in press. enolide orthoacetates and two related acetate esters.' (2) This investigation was supported by grants from t h e National I n The observation that hellebrigenin 3-acetate showed stitutes of Health (HE-12957 and CA-11718) and the American Cancer significant activity in vivo against the Walker intraSociety (T-275) and a contract with Chemotherapy, National Cancer Institute, National Institutes of Health (PH-43-64-551). C. W. 9. was a muscular carcinosarcoma 256 in rats stimulated further
N I H Postdoctoral Fellow, 1967-1969. (3) Author t o whom inquiries should be directed: Department of Chemist r y , University of Virginia. (4) 6. M . Kupchan, T r a n s . N . Y . A c a d . Sei., 82, 85 (1970). ( 5 ) Fruits of B . abyssinica were collected in Ethiopia, J a n 1968. The authors acknowledge with thanks receipt of the dried plant material from Dr. Robert E . Perdue, U. 8. Department of Agriculture, Beltsville, Md., in accordance with the program developed with USDA by the Cancer Chemotherapy National Elervice Center (CCNSC).
(6) Cytotoxicity was assayed under t h e auspices of the CCNSC and the procedures were those described in Cancer Chemother. Rep., 26, 1 (1962). Cytotoxicity was also assayed b y differential agar diffusion by Professor D. Perlman, University of Wisconsin: cf. D. Perlman and J. L. Schwarta, J . Pharm. Sci., 68, 633 (1969). (7) S. M. Kupchan, R. J. Hemingway, and J. C. Hemingway, J . O w . Chem., 34, 3894 (1969).
